Microbiology Exam #2

Most of them are bacteriostatic. Aminoglycosides (e.g. streptomycin) are bactericidal (too many wrong proteins made). Resistance methods are varied.

Describe antibiotics that interfere with protein synthesis

Quinolones bind to DNA gyrase and prevent the ligation step, leading to lots of double-stranded breaks, which hinders DNA replication/transcription. Many side effects because human mitochondria evolved from bacteria. Resistance develops via mutation in GryA/GryB and efflux pump

Describe antibiotics that target nucleic acids and resistance mechanisms

Sporulation is regulated by a sigma factor cascade (crisscross regulation). Intercellular communication across the polar septum to make sure events in the mother cell/endospore happen at the right times relative to each other.

Describe gene regulation during the sporulation process

Introducing a defined mutation into a gene at a precise site 1. Clone the plasmid and denature 2. Add synthetic oligonucleotide with one base mismatch 3. Extend single strand with DNA polymerase 4. Digest with Dnpl to cleave template DNA 5. Transformation and selection

Describe site-directed mutagenesis

Sulfa drugs (sulfonamide) interfere with the synthesis of folic acid (vitamin B9) which bacteria need. Mimic PABA (compound in bacteria) and compete for an enzyme that synthesizes folic acid. Resistance mechanism: synthesizing large amounts of PABA

Describe sulfa drugs

1. It can be catabolized by the TCA cycle and generate additional electron carriers for the electron transport chain 2. It can feed into a fermentation pathway

2 fates of pyruvate generated during glucose catabolism

Engineered auxotrophy - survival of the engineered microbe relies on supplementation with essential compound Induced lethality - a 'kill switch' is activated by an inducer that is added to or present in the natural environment (e.g. certain sugars, high temperature)

2 types of biocontainment strategies

1. Extract a plasmid 2. Cleave plasmid and foreign DNA with EcoR1 endonuclease 3. Insert and anneal the new strand to the plasmid with DNA ligase

3 step process for making recombinant DNA

1. Look at cells under a light microscope to see if they move 2. Swarming motility/soft agar assay - plate cells on semi solid medium that will hold non-motile bacteria in place but motile bacteria can move around. A small dot on a plate indicates non-motile bacteria and a big splotch indicates the bacteria were motile 3. Capillary assay - take a straw with a substance in it and submerge it in a culture and see how the bacteria react

3 ways to determine if bacteria are motile or not

1. Modify genes to create proteins with novel functions 2. Identify existing genes that encode proteins with desired functions 3. Precisely control protein levels 4. Biocontainment

4 design challenges of synthetic biology

In theory you can get 3 ATP per NADH and 2 ATP per FADH2

ATP yield of NADH and FADH2?

1. Very specific to strain 2. Phages can multiply at the site of infection so the initial dose can be lower than the therapeutic dose and you may only need a single dose 3. The phage could evolve to evade resistance mechanisms 4. Phages can bind to surface virulence factors. Phage resistance is most easily achieved by modifying or deleting the phage attachment site, which could prevent virulence. Sets up a genetic tradeoff between virulence and phage/antibiotic resistance You would need rapid screening though

Advantages of phage therapy?

1. DNA replicates and extends into an axial filament 2. Septum forms near one pole separating forespore from mother cell. Each gets a chromosome 3. Mother cell engulfs the forespore, surrounding it with a second membrane. 4. The endospore produces a PG cortex between the inner and outer membranes. 5. The mother cell produces a spore coat 6. Dipicolonic acid is synthesized and calcium is incorporated into the spore coat 7. Programmed cell death for the mother cell, releases the spore

Bacillus subtilis sporulation process

Bacteriostatic - prevents growth Bactericidal - actually kills the bacteria

Bacteriostatic vs. bactericidal??

Rifampin binds to bacterial RNA polymerase and stops transcription by blocking RNA from exiting the polymerase

Describe RNA synthesis inhibitor antiobiotics

1. Beta-lactams target the cell wall. Resistant bacteria primarily produce beta-lactamase, which breaks the 4-member ring in beta-lactam. To fight resistance, clauvanic acid can bind to and inhibit beta-lactamase 2. Vancomycin binds to the D-Ala-D-Ala terminal end and prevents transglycosylation/transpeptidation. Typically used as a drug of last resort

Describe antibiotics targeting the cell wall and resistance mechanisms

Chemotaxis

Cell movement towards or away from a chemical gradient

Phototaxis

Cell movement towards or away from light

Aerotaxis

Cell movement towards or away from oxygen

Magnetotaxis

Cell orientation in a magnetic field (like Earth's, to determine which way is up and to help the bacterium find nutrients and adjust its depth in an aquatic environment)

Logic gates are a mechanism to control protein production levels 1. "Buffer gate" - amplifies signals (inducer molecule -> output molecule) 2. "NOT gate" - represses protein production (repressor molecule -> output molecule) 3. "OR gate" - involves several genes (alternative input molecules driving production of alternative proteins, either of which can activate expression of the output gene)

Describe the 3 different logic gates and why they are used?

Polymyxin is a polypeptide antibiotic - binds to inner and outer membrane of bacteria and destroys the inner membrane

Describe the antibiotic targeting the cell membrane

1. MCPs (methyl-accepting chemotaxis proteins AKA transmembrane chemoreceptors) are methylated 2. A methylated MCP stimulate CheA kinase autophosphorylation. 2. The phosphoryl group is transferred to CheY (single domain response regulator). 3. CheY~P interacts with the flagellar switch (FliM). Motion changes from CCW to CW, and causes a tumble. 4. CheZ stimulates CheY~P dephosphorylation such that the switch in flagellar rotation is transient

Describe the chemotaxis excitation pathway

MCPs are continuously methylated and demethylated such that a basal level is maintained. The methylation state of the MCPs regulates their ability to simulate CheA autophosphorylation.

Describe the chemotaxis pathway in the absence of a chemical gradient

NADH oxidation is typically coupled to the reduction of pyruvate (or one of its derivatives) which generates fermentation products. To compensate for less ATP produced than respiration, microbes consume large amounts of substrate and excrete large amounts of products

Describe the fermentation process

First it penetrates the prey cell's outer membrane and cell wall and into the periplasm. Then, the host cell rounds up giving the bacteria space to grow. As the bacteria replicates its DNA, it eats the host cell. When the host cell is all gone, the bacteria septates into several new cells they develop, and then leave the cell through lysis.

Describe the predatory development program of Bdellovibrio bacteriovorus

Multiple checkpoints, rather than a simple histidine kinase -> response regulator pathway

Describe the process of sporulation activation

1. Rhizobia binds to the root hair of the plant, secretes Nod factors (lipooligosaccharides) 2. The Nod factors cause the root hair to curl and trap a microcolony of Rhizobia. Plant cells in the inner cortex initiate cell division to make the nodules on the roots. 3. Root hair cell wall invaginates and extends via tip growth down the root hair. 4. Infection threads deliver the bacteria into the nodules 5. Bacteria enter the cytoplasm of the nodule cells. 6. The bacteria differentiate into the large Y-shaped bacterioids and begin fixing nitrogen

Describe the process of the invasion of legume plants by rhizobia

When starved, vegetative mycelium form aerial hyphae. The aerial hyphae elongate (via FtsZ), septate, then form spores that can start other colonies.

Describe the sporulation process of the filamentous bacterium Streptomyces

When a cell is exposed to the same stimulus for a long time, the adaptation pathway causes cells to stop responding to it. If a cell is continuously exposed to increasing attractant concentrations, reduced CheA autophosphorylation decreases CheB~P level (more slowly than CheY~P level). CheB methylesterase activity is therefore reduced, so the MCPs become more highly methylated. Increased methylation increases the ability of the MCPs to stimulate CheA autophosphorylation, counteracting the inhibitory effect of the attractant. As CheA autokinase activity increases, CheY~P concentration returns to its pre-stimulus value and the flagellar motor resumes its pre-stimulation CW to CCW switching ratio (random walk)

Describe when the adaptation pathway is activated in chemotaxis and what the process is

Break the genome up into DNA fragments, sequence all of the fragments, then use a computer to piece them together

Describe whole genome shotgun sequencing

Chemoorganotrophs use organic chemicals as their energy source. Chemolithotrophs use inorganic chemicals

Difference between chemoorganotrophs and chemolithotrophs?

Not the concentration of the chemical itself but temporal changes in concentration. Bacteria reset their signaling system frequently so they always know what the concentration is relative to a few seconds ago.

In chemotaxis, what are cells actually sensing?

2 ATP, 2 NADH, 2 pyruvate

Emden-Meyerhof-Parnas (EMP) pathway net yield

1 ATP, 1 NADPH, 1 NADH, 1 pyruvate. The second part of the pathway is the same as EMP.

Entner-Doudoroff (ED) pathway net yield and how is it similar to glycolysis (EMP)?

1. Smooth swimming while flagella are rotating CCW (run) 2. CW motion: bundle of flagella is no longer stable and going in different directions (tumble) 3. As cell begins to move in one direction, the flagella form a thermodynamically stable bundle of flagella behind the cell (CCW rotation) (run) Reference: Lecture 10 Slide 21

Explain process of run and tumble motion

Fla-: no flagellum, can't swim, no chemotaxis Mot-: flagellum, can't swim, no chemotaxis Che-: flagellum, can swim, no chemotaxis

Explain the 3 phenotypes Fla- Mot- and Che- in bacteria

1. Euk. flagella undulates like a snake inside its cellular membrane but the bacterial flagellum works like a power boat with a rotary motor turning a helical propellor. 2. Euk. flagellum uses ATP as a source of energy whereas bacterial flagellum uses electromotive gradient of H+ or Na+ across the cell membrane 3. 20 different proteins in bacterial flagellum, over 200 in euk. flagellum 4. Bacterial flagellum is ~10µm long while euk. flagellum is 100µm long

Four ways bacteria flagella are different from eukaryotic flagella

substrate dehydrogenase, mobile electron carrier, terminal oxidase

Functional components of the electron transport chain

Artemisin is the primary therapy for malaria. It's usually extracted from the wormwood plant but that's expensive. Scientists have modified the yeast mevalonate pathway and introduced an enzyme from the wormwood plant to make artemisinic acid, which is secreted by the yeast. Artemisinic acid can be used to synthesize artemisinin.

How are people bioengineering a cure for malaria?

1. Natural product discovery - especially through finding ways to culture the "unculturables" 2. Continued modification of existing antibiotics 3. Reevaluating old antibiotics that were discontinued due to side effects 4. Phage therapy???

How are people trying to come up with novel antibiotics?

Narrow or limited spectrum antibiotics may be better since they're less likely to disrupt the patient's natural microbiota, less likely to be overprescribed/inappropriately used, may be harder to develop resistance, and they're easier to find

How are we considering spectrum of antibiotics now?

With a glyoxylate shunt: requires 2 molecules of acetyl-CoA, bypasses decarboxylation steps. Net yield for 2 acetates: 2 NADH, 1 FADH2

How can cells grow on acetate instead of glucose?

Polar flagella can rotate clockwise or counterclockwise so cells can move backward or forwards. The hook tends to buckle when cells move forwards, causing cells to turn.

How do bacteria with polar flagella change directions?

Through sulfate reduction. In sulfate reducing bacteria, oxidation of a carbon source is coupled with the reduction of sulfate. Sulfate, the terminal electron acceptor, is reduced to hydrogen sulfide. Hydrogen sulfides can precipitate metal contaminants via the formation of metal sulfides.

How does Desulfovibrio vulgaris detoxify Cr(VI)?

1. Attach complementary primers to the slide, spatially separated clonal molecules. 2. Add fragments of DNA (single-stranded) from your sample. Wash away any strands that don't attach to the primers. 3. Do PCR such that the strands can replicate to form clusters of identical strands all attached to the slide. 4. Add fluorescent terminator ddNTPs. Produce an image of the slide. Remove the terminator-fluorescent group from the ddNTPS. Repeat.

How does Illumina sequencing work?

1. Denature DNA into single strands 2. Add regular nucleotides plus a small amount of nucleotides with fluorescent labels missing the 3' OH end (strand won't be able to replicate if a nucleotide without a 3' OH end is incorporated) 3. The DNA will start to replicate - size of the fragments corresponds to the time at which the missing 3' OH nucleotide is incorporated 4. Fragments are put through a gel and you can read the sequence by reading the labels corresponding to GATC in order of length of the strand

How does Sanger sequencing work?

1. Add protease to digest protein (AKA break down into peptides) 2. Peptides separated by column chromatography 3. Effluent passed into mass spectrometer 4. Mass calculations provide molecular weight of each peptide 5. Protein is determined by sum of its peptide masses 6. Peptides quantified by heights of peak signals

How does protein mass spectrometry work?

F1FoATP synthase is a highly conserved protein complex made of two parts: 1. Fo: embedded in the mitochondrial membrane, pumps protons 2. F1: protrudes in the cytoplasm, generates ATP Fo (the C ring) rotates 1/3 of a turn relative to F1 for every 3 protons pumped, generating 1 ATP

How does the F1FoATP synthase work?

The flagellum is constructed from base to tip with linear assembly. MS ring is constructed first in the cytoplasmic membrane, then the C ring (controls direction of rotation) beneath it in the cytoplasm, then the MotA/MotB proton channel motors which connect to the PG layer. The PG layer is degraded by a flagellum specific muramidase to make way for the P ring. The capping complex is always attached at the distal end, promoting an efficient assembly process. All axial proteins including rod, hook, and flagellin subunits are transported in a partially unfolded state by a Type III secretion apparatus (powered by ATP hydrolysis) which binds underneath the base of the flagellum. Ref: Slides 12-14 in Lecture 10

How is the flagellum constructed?

Just before elongation starts, the capping complex (HAP2) forms a cap on top. The cap is a pentameter of the HAP2 protein with the shape of a 5-leg table. It binds at the distal end of the flagellar filament and promotes the efficient self-assembly of flagellin subunits transported to the distal end through the narrow central channel of the flagellum.

How is the flagellum elongated?

Net yield: 2 NADPH. Like ED, begins by forming 6-phosphogluconate. Key intermediate: ribulose-5-phosphate. It produces a series of sugars with 3-7 carbons each, and can feed into EMP or serve as precursors for DNA, RNA, or aromatic amino acids

Pentose Phosphate pathway (PPP) net yield. How is it similar to the ED pathway? What key intermediate does it produce and why is it important?

1. Extract total RNA 2. Enrich RNA 3. Fragment mRNA 4. Convert to cDNA 5. Library construction for Illumnia sequencing 6. Sequencing

mRNA-Seq process

1. Extract proteins from sample 2. Separate intact or chopped-up proteins by their physical properties 3. Analyze by mass spectrometry

Proteomics workflow?

38 ATP

Theoretical ATP yield of complete oxidation of glucose?

2 ATP, 8 NADH, 2 FADH2

Tricarboxylic acid cycle (TCA) (AKA citric acid cycle or Krebs cycle) net yield for 2 pyruvates

16S sequencing Pros: Can identify species level composition of microbial communities, doesn't rely on gene annotations, routinely performed, cost-effective Cons: No information about metabolic potential, can't differentiate between different strains of the same species Whole genome shotgun sequencing Pros: Can identify metabolic activities (functional genomics), and different strains within a population Cons: Limited genome assembly, relies heavily on reference sequences, more expensive and computationally demanding than 16S sequencing

Two key approaches to metagenomics

Polar: one polar flagellum on the bacterium Peritrichous: several flagella all over the bacterium, combine to form a helical bundle when the cell is swimming

Two types of flagellar arrangements

Broad spectrum - effective across a wide range of Gram+ and Gram- bacteria Narrow spectrum - effective against a wide range of either Gram+ OR Gram- bacteria Limited spectrum - effective against a single organism or disease

What are the 3 different spectrums of action of antibiotics?

1. Glycolysis (AKA the Embden-Meyerhof-Parnas (EMP) pathway) (most common) 2. Entner-Doudoroff (ED) pathway 3. Pentose phosphate pathway (PPP)

What are the 3 routes bacteria use to catabolize glucose?

1. Drug target modification 2. Drug modification/inactivation 3. Barrier to entry 4. Drug efflux pump

What are the 4 mechanisms of antibiotic resistance?

ATP, GTP, NADH

What are the energy carrying molecules in metabolism?

1. Attachment 2. Growth 3. Detachment

What are the three major stages in the life cycle of a biofilm?

High temperatures (100C), ionizing radiation, chemical solvents, detergents and hydrolytic enzymes, and can remain dormant for long periods of time.

What can spores survive?

Flagella for aqueous environments, pili for moving around on solid surfaces

What do bacteria use to move around in aqueous environments and on solid surfaces

CheB is a response regulator. Phosphorylation activates its methylesterase activity (results in demethylation of MCP).

What does CheB do?

Complex mixture of exopolysaccharies, proteins, and eDNA

What does a biofilm consist of?

Carries electrons from NADH and FADH2 (generated during glycolysis, TCA cycle) to a terminal electron acceptor.

What does the electron transport chain do?

"Proton motive force" that drives protons across the membrane. The free energy from the PMF is used to generate ATP via ATP synthase (chemiosmosis)

What does the electron transport chain generate?

An increase in the concentration of an attractant decreases CheA autophosphorylation, and an increase in the concentration of a repellant increases CheA autophosphorylation (excites the pathway)

What happens to CheA autophosphorylation if the concentration of an attractant increases? What will happen if the concentration of a repellant increases?

It divides into a stalked cell and a swarmer cell (with flagella instead of a stalk). This is caused by a gradient of DivK~P along the length of the cell. Where DivK~P is the lowest, PleC phosphatase forms (flagellum side). When DivK~P is at its highest, DivJ kinase forms (stalk side)

What happens when Caulobacter crescentus divides?

Minimum inhibitory concentration (MIC) is the minimum concentration for an antibiotic to work. Multiple doses of antibiotics keep its concentration above MIC in the body

What is MIC?

Therapeutic index (TI) is the ratio between the toxic dose (to humans) and the therapeutic dose (to eliminate bacteria)

What is TI?

In the presence of a chemical gradient, the cell tumbles less frequently when it goes toward that gradient

What is a "biased random walk"?

In a homogenous environment, the cell tumbles at random intervals and its motion isn't biased in any direction

What is a "random walk" in cell motion?

Reactions that build complex molecules from smaller ones

What is anabolism?

Breakdown of complex molecules into smaller ones (provides energy for anabolism)

What is catabolism?

Use genomic data to determine similarities and differences between genomes of different organisms.

What is comparative genomics?

Mutation of a gene at several nonspecific sites coupled to selection of mutants with desired functions

What is directed evolution?

Use genomic data to predict gene and protein functions and interactions (focus is on dynamic properties of gene products)

What is functional genomics?

The process of isolating potentially useful, novel genes from the environment without culturing the parent organism Process: 1. Collect DNA samples from different environments 2. Construct genomic library 3. Transform host cells and plate on selective media 4. Screen library for reactive colonies

What is gene mining?

Analysis of microbial communities in their natural environments using genomic techniques - advantageous because we can't culture 99% of microbial life

What is metagenomics?

Mutated genes are cloned into a phage capsid gene and "displayed" on the phage surface for subsequent selection. Phages can then be "biopanned" for phage-display proteins with desired binding affinities by putting phages on a plate coated with a target to see which phages have proteins that bind to the target.

What is phage display?

Simultaneously measuring all the proteins in a cell or organism

What is proteomics?

When a phosphate group is transferred from a high-energy substrate (food) to ADP, forming ATP

What is substrate-level phosphorylation?

The redesign or construction of biological systems through genetic engineering

What is synthetic biology?

Simultaneously measuring the transcription of every gene in a cell (i.e. all of the cell's mRNA molecules)

What is transcriptomics?

c-di-GMP

What signal molecule regulates biofilm transition? (as well as processes in motility, virulence, and cell cycle progression)

Rapid drug susceptibility testing (DST) is PCR based testing of patients for antibiotic resistant bacteria. It reduces treatment failures, transmission of drug-resistant pathogens, allows for appropriate treatment of drug-resistant pathogens

What's rapid DST?

At the poles of the cell

Where are chemoreceptors (MCPs) clustered?

Considered secondary metabolites. May have initially evolved for cell-to-cell signaling or interspecies competition. The producer has mechanisms to avoid suicide.

Where do antibiotics come from?

1. Expensive 2. Supposed to be used sparingly 3. Become obselete after resistance is widespread 4. Often have side effects

Why don't companies focus on making antibiotics anymore? 4 reasons

Proteins can be metabolically taxing and toxic to the host. Proteins can aggregate or be degraded by intracellular proteases

Why is it important to control the production of a foreign protein?